U.S. patent application number 12/690579 was filed with the patent office on 2010-07-29 for systems and methods for selective detection and imaging in coherent raman microscopy by spectral-temporal excitation shaping.
Invention is credited to Christian Freudiger, Wei Min, Xiaoliang Sunney Xie.
Application Number | 20100188496 12/690579 |
Document ID | / |
Family ID | 42153726 |
Filed Date | 2010-07-29 |
United States Patent
Application |
20100188496 |
Kind Code |
A1 |
Xie; Xiaoliang Sunney ; et
al. |
July 29, 2010 |
SYSTEMS AND METHODS FOR SELECTIVE DETECTION AND IMAGING IN COHERENT
RAMAN MICROSCOPY BY SPECTRAL-TEMPORAL EXCITATION SHAPING
Abstract
A microscopy imaging system is disclosed that includes a light
source system, a spectral shaper, a modulator system, an optics
system, an optical detector and a processor. The light source
system is for providing a first train of pulses and a second train
of pulses. The spectral shaper is for spectrally modifying an
optical property of at least some frequency components of the
broadband range of frequency components such that the broadband
range of frequency components is shaped producing a shaped first
train of pulses to specifically probe a spectral feature of
interest from a sample, and to reduce information from features
that are not of interest from the sample. The modulator system is
for modulating a property of at least one of the shaped first train
of pulses and the second train of pulses at a modulation frequency.
The optical detector is for detecting an integrated intensity of
substantially all optical frequency components of a train of pulses
of interest transmitted or reflected through the common focal
volume. The processor is for detecting a modulation at the
modulation frequency of the integrated intensity of substantially
all of the optical frequency components of the train of pulses of
interest due to the non-linear interaction of the shaped first
train of pulses with the second train of pulses as modulated in the
common focal volume, and for providing an output signal for a pixel
of an image for the microscopy imaging system.
Inventors: |
Xie; Xiaoliang Sunney;
(Lexington, MA) ; Freudiger; Christian; (Boston,
MA) ; Min; Wei; (Cambridge, MA) |
Correspondence
Address: |
GAUTHIER & CONNORS, LLP
225 FRANKLIN STREET, SUITE 2300
BOSTON
MA
02110
US
|
Family ID: |
42153726 |
Appl. No.: |
12/690579 |
Filed: |
January 20, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61147190 |
Jan 26, 2009 |
|
|
|
Current U.S.
Class: |
348/79 ;
348/E7.085 |
Current CPC
Class: |
G01N 21/65 20130101;
G02B 21/082 20130101; G01N 2021/653 20130101; G01J 3/44 20130101;
G01J 3/10 20130101; G02B 21/002 20130101; G01J 2003/1282 20130101;
G01N 2021/655 20130101; G01N 2201/0675 20130101 |
Class at
Publication: |
348/79 ;
348/E07.085 |
International
Class: |
H04N 7/18 20060101
H04N007/18 |
Goverment Interests
[0002] This invention was made with government support under
DE-FG02-07ER15875 awarded by the U.S. Department of Energy,
OD000277 awarded by the National Institutes of Health, and 0649892
awarded by the National Science Foundation. The government has
certain rights to the invention.
Claims
1. A microscopy imaging system comprising: a light source system
for providing a first train of pulses including a first broadband
range of frequency components, and a second train of pulses
including a second optical frequency such that a set of differences
between the first broadband range of frequency components and the
second optical frequency is resonant with a set of vibrational
frequencies of a sample in the focal volume, wherein the second
train of pulses is synchronized with the first train of pulses; a
spectral shaper for spectrally modifying an optical property of at
least some frequency components of the broadband range of frequency
components such that the broadband range of frequency components is
shaped producing a shaped first train of pulses to specifically
probe a spectral feature of interest from a sample, and to reduce
information from features that are not of interest from the sample;
a modulator system for modulating a property of at least one of the
shaped first train of pulses and the second train of pulses at a
modulation frequency to provide a modulated train of pulses; an
optics system for directing and focusing the shaped first train of
pulses and the second train of pulses as modulated toward a common
focal volume; an optical detector for detecting an integrated
intensity of substantially all optical frequency components of a
train of pulses of interest transmitted or reflected through the
common focal volume; and a processor for detecting a modulation at
the modulation frequency of the integrated intensity of
substantially all of the optical frequency components of the train
of pulses of interest due to the non-linear interaction of the
shaped first train of pulses with the second train of pulses as
modulated in the common focal volume, and for providing an output
signal for a pixel of an image for the microscopy imaging
system.
2. The microscopy imaging system as claimed in claim 1, wherein
only one of the shaped train of laser pulses or the second train of
pulses is modulated at the modulation frequency to provide the
modulated train of pulses such that the other of the shaped train
of laser pulses and the second train of pulses remains a
non-modulated train of pulses; wherein the optical detector detects
the integrated intensity of substantially all optical frequency
components of the non-modulated train of pulses transmitted or
reflected through the common focal volume by blocking the modulated
train of pulses; and wherein the processor detects a modulation at
the modulation frequency of the integrated intensity of
substantially all of the optical frequency components of the
non-modulated train of pulses due to the non-linear interaction of
the modulated train of pulses with the non-modulated train of
pulses in the common focal volume.
3. The microscopy imaging system as claimed in claim 2, wherein
said optical property of either the shaped first train of pulses or
the second train of pulses that is modulated is amplitude.
4. The microscopy imaging system as claimed in claim 2, wherein
said optical property of either the shaped first train of pulses or
the second train of pulses that is modulated is polarization and
wherein said system further includes a polarization analyzer.
5. The microscopy imaging system as claimed in claim 1, wherein
said spectral shaper and said modulator system are included in the
same device.
6. The microscopy imaging system as claimed in claim 2, wherein
said broadband range of frequency components includes a range of
frequency components of at least 0.5 nm.
7. The microscopy imaging system as claimed in claim 1, wherein
said broadband range of frequency components is discontinuous.
8. The microscopy imaging system as claimed in claim 2, wherein
said second train of pulses also has a broadband range of frequency
components.
9. The microscopy imaging system as claimed in claim 2, wherein the
modulation frequency is at least 100 kHz.
10. The microscopy imaging system as claimed in claim 2, wherein
said imaging system employs stimulated Raman spectroscopy as a
contrast mechanism, and wherein the first train of pulses provides
a pump beam, and the second train of pulses is modulated by the
modulator system at the modulation frequency to provide one of a
pump beam and a Stokes beam such that a Raman loss is detected at
the signal processor at the modulation frequency.
11. The microscopy imaging system as claimed in claim 2, wherein
said imaging system employs stimulated Raman spectroscopy as a
contrast mechanism, and wherein the first train of pulses provides
a pump beam, and the second train of pulses is modulated by the
modulator system at the modulation frequency to provide one of a
pump beam and a Stokes beam such that a Raman gain is detected at
the signal processor at the modulation frequency.
12. The microscopy imaging system as claimed in claim 1, wherein
said processor detects a modulation of the integrated intensity of
substantially all of the optical frequency components of a train of
anti-Stokes pulses due to the non-linear interaction of the shaped
first train of pulses with the second train of pulses as modulated
in the common focal volume.
13. The microscopy imaging system as claimed in claim 1, wherein
said system employs two-photon absorption as a contrast mechanism
in which one photon from the first train of pulses and a second
photon from the second train of pulses are simultaneously
absorbed.
14. The microscopy imaging system as claimed in claim 2, wherein
said spectral shaper includes one of a spatial light modulator, a
dazzler system, a multiplex electro-optic modulator, a multiplexed
electro-acoustic modulator, or an acousto-optic tunable filter.
15. The microscopy imaging system as claimed in claim 2, wherein
said spectral shaper differently modifies a polarization of
different frequency components of the broadband range of frequency
components of the first train of pulses.
16. The microscopy imaging system as claimed in claim 2, wherein
said spectral shaper differently modifies an amplitude of different
frequency components of the broadband range of frequency components
of the first train of pulses.
17. The microscopy imaging system as claimed in claim 2, wherein
said spectral shaper includes a spectral dispersion unit and a
polarization spatial light modulator, and wherein said modulator is
a polarization modulator, and wherein said system further includes
a polarization analyzer that is positioned before or after the
modulator.
18. A method of performing microscopy imaging using frequency
modulation comprising the steps of: providing a first train of
pulses at including a first broadband range of optical frequency
components; providing a second train of pulses including a second
optical frequency such that a set of differences between the first
broadband range of frequency components and the second optical
frequency is resonant with a set of vibrational frequencies of a
sample in the focal volume, wherein the second train of pulses is
synchronized with the first train of pulses; spectrally modifying
an optical property of at least some frequency components of the
first broadband range of frequency components to provide a shaped
first train of pulses that is shaped to specifically probe a
spectral feature of interest from a sample, and to reduce
information from features that are not of interest from the sample;
modulating an optical property of one of the shaped first train of
pulses and the second train of pulses at a modulation frequency to
provide a modulated train of pulses and providing the other of the
shaped first train of pulses and the second train of pulses as a
non-modulated train of pulses; directing and focusing the modulated
train of pulses and the non-modulated train of pulses toward a
common focal volume; detecting an integrated intensity of
substantially all optical frequency components of the other of the
modulated train of pulses and the non-modulated train of pulses
transmitted or reflected through the common focal volume by
blocking the modulated train of pulses; detecting a modulation at
the modulation frequency of the integrated intensity of
substantially all of the optical frequency components of the
non-modulated train of pulses due to the non-linear interaction of
the modulated train of pulses with the non-modulated train of
pulses in the common focal volume; and providing the detected
modulation as the signal for a pixel of an image for a microscopy
imaging system.
19. The method as claimed in claim 18, wherein said step of
spectrally modifying an optical property of at least some frequency
components and the step of modulating an optical property of one of
the shaped first train of pulses and the second train of pulses to
provide a modulated train of pulses is performed by the same
device.
20. The method as claimed in claim 18, wherein said method further
includes the steps of: further spectrally modifying an optical
property of at least further frequency components of the broadband
range of frequency components of the first train of pulses to
provide a further shaped first train of pulses to specifically
probe a spectral feature from a sample that interferes with the
spectral feature of interest from the sample; subtracting the
detected modulation of the integrated intensity of substantially
all of the optical frequency components of the non-modulated train
of pulses due to the non-linear interaction of the further shaped
first train of pulses and the second train of pulses in the focal
volume from the detected modulation of the integrated intensity of
substantially all of the optical frequency components of
non-modulated train of pulses due to the non-linear interaction of
the originally shaped first train of pulses and the second train of
pulses in the focal volume; and providing the difference as the
signal for a pixel of an image for the microscopy imaging
system.
21. The method as claimed in claim 20, wherein said step of further
spectrally modifying an optical property of at least further
frequency components of the broadband range of frequency components
of the first train of pulses is performed for an entire scan area
prior to the step of subtracting the detected modulation of the
integrated intensity of substantially all of the optical frequency
components obtained thereby from the detected modulation of the
integrated intensity of substantially all of the optical frequency
components of non-modulated train of pulses.
22. The method as claimed in claim 21, wherein said step of further
spectrally modifying an optical property of at least further
frequency components of the broadband range of frequency components
of the first train of pulses and the step of subtracting the
detected modulation of the integrated intensity of substantially
all of the optical frequency components obtained thereby from the
detected modulation of the integrated intensity of substantially
all of the optical frequency components of non-modulated train of
pulses are performed for one pixel prior to laser scanning to the
next pixel.
23. The method as claimed in claim 18, wherein said step of
spectrally modifying an optical property of at least some frequency
components of the broadband range of frequency components of the
shaped first train of pulses involves amplitude modulation.
24. The method as claimed in claim 18, wherein said step of
spectrally modifying an optical property of at least some frequency
components of the broadband range of frequency components of the
shaped first train of pulses involves polarization modulation.
25. The method as claimed in claim 18, wherein said method includes
the steps of providing different spectral masks at different
modulation frequencies, as well as the steps of detecting multiple
trains of pulses of interest using multiple lock-in detectors tuned
to the different modulation frequencies such that a plurality of
species may be probed at the same time.
26. A method of performing microscopy imaging comprising the steps
of: a) providing a first train of pulses at including a first
broadband range of optical frequency components; b) providing a
second train of pulses including a second optical frequency such
that a set of differences between the first broadband range of
frequency components and the second frequency component is resonant
with a set of vibrational frequencies of a sample in the focal
volume, wherein the second train of pulses is synchronized with the
first train of pulses; c) spectrally modifying an optical property
of at least some frequency components of the first broadband range
of frequency components such that the first train of pulses is
shaped to provide a shaped first train of pulses to specifically
probe a spectral feature of interest from a sample; d) modulating a
property of one of the shaped first train of pulses and the second
train of pulses at a modulation frequency to provide a modulated
train of pulses and to provide the other of the shaped first train
of pulses and the second train of pulses as a non-modulated train
of pulses; e) directing and focusing the modulated train of pulses
and the non-modulated train of pulses toward a common focal volume;
f) detecting an integrated intensity of substantially all optical
frequency components of the non-modulated train of pulses at a
modulation frequency transmitted or reflected through the common
focal volume by blocking the modulated train of pulses; g)
detecting a modulation at the modulation frequency of the
integrated intensity of substantially all of the modulated train of
pulses due to the non-linear interaction of the modulated train of
pulses with the non-modulated train of pulses in the common focal
volume; h) further spectrally modulating an optical property of at
least some frequency components of the first broadband range of
frequency components such that the first train of pulses is
negatively shaped to provide to provide a negatively shaped first
train of pulses to specifically probe a spectral feature from a
sample that interferes with the spectral feature of interest from
the sample; i) modulating a property of one of the negatively
shaped first train of pulses and the second train of pulses at a
modulation frequency to provide a further modulated train of pulses
to provide the other of the shaped first train of pulses and the
second train of pulses as a non-further modulated train of pulses;
j) directing and focusing the further modulated train of pulses and
non-further modulated train of pulses toward a common focal volume;
k) detecting an modulation of an integrated intensity of
substantially all optical frequency components of
non-further-modulated train of pulses and the further modulated
train of pulses at a modulation frequency transmitted or reflected
through the common focal volume by blocking the further modulated
train of pulses; l) subtracting the modulation of the integrated
intensity of substantially all of the optical frequency components
obtained from the modulation of the integrated intensity of
substantially all of the further modulated train of pulses due to
the non-linear interaction of the further modulated train of pulses
and the non-further modulated train of pulses in the common focal
volume to obtain a difference signal; and m) providing an image for
the microscopy imaging system responsive to the difference
signal.
27. The method as claimed in claim 26, wherein each of the steps
a)-m) is performed for each pixel in a microscopy imaging system
prior to each of the steps a)-m) is performed for another
pixel.
28. The method as claimed in claim 26, wherein each of the steps
a)-g) is performed for each pixel in a microscopy imaging system
prior to each of the steps h)-m) is performed for each pixel.
Description
PRIORITY
[0001] The present application claims priority to U.S. Provisional
Patent Application Ser. No. 61/147,190 filed Jan. 26, 2009.
BACKGROUND
[0003] The invention generally relates to vibrational microscopy
and imaging systems, and relates in particular to vibrational
imaging systems employing coherent Raman scattering.
[0004] Conventional vibrational imaging techniques include, for
example, infrared microscopy, spontaneous Raman microscopy, and
coherent anti-Stokes Raman scattering microscopy.
[0005] Infrared microscopy, which generally involves directly
measuring the absorption of vibrational excited states in a sample,
is limited by poor spatial resolution due to the long wavelength of
infrared light, as well as by a low penetration depth due to a
strong infrared light absorption by the water in biological
samples.
[0006] Raman microscopy records the spontaneous inelastic Raman
scattering upon a single (ultraviolet, visible or near infrared)
continuous wave (CW) laser excitation. Raman microscopy has
improved optical resolution and penetration depth as compared to
infrared microscopy, but the sensitivity of Raman microscopy is
rather poor because of the very low spontaneous Raman scattering
efficiency; a Raman scattering cross section is typically on the
order of 10.sup.-30 cm.sup.2. This results in long averaging times
per image, which limits the biomedical applications of Raman
microscopy.
[0007] Coherent anti-Stokes Raman (CARS) microscopy systems provide
increased scattering signal from a sample due to coherent
excitation. CARS microscopy systems use two pulsed excitation laser
beams (pump and Stokes beams) with a frequency difference that
matches the molecular vibration frequency of the chemical species
to be imaged. As a result of interaction of the chemical species to
be imaged with the difference frequency between the pump and Stokes
beams, new illumination is generated at the sample at the
anti-Stokes frequency, which is detected as the output signal in
CARS microscopy. Imaging speeds up to video-rate have been achieved
from highly resonant samples.
[0008] The CARS process, however, also excites a high level of
background from the vibrationally non-resonant specimen. Such a
non-resonant background not only distorts the CARS spectrum of the
resonant signal from dilute samples but also carries the laser
noise, significantly limiting the application of CARS microscopy on
both spectroscopy and sensitivity perspectives. Various techniques
have been developed to suppress this background, as disclosed, for
example, in U.S. Pat. Nos. 6,798,507 and 6,809,814, but such
systems each provides an anti-Stokes signal that is at least
somewhat reduced by the background suppression.
[0009] Moreover, the specificity of the anti-Stokes signals for
certain target species is limited because many chemical species may
have a vibrational response at multiple frequencies. For example,
FIG. 1 shows at 10 a Raman spectrum for the bioactive molecule
adenosine triphosphate (ATP), the chemical formula for which is
shown at 12 in FIG. 2. Note that because ATP has many different
types of atomic bonds, it has several Raman active peaks that
together provide a characteristic vibrational signature of the
molecules.
[0010] The specificity is limited since many different chemical
species (e.g., one target species and one non-target species) may
have some of the same bonds (e.g., O--H) that provide the same
vibrational response at the anti-Stokes frequency to the excitation
illumination, making distinguishing between the two chemical
species difficult or impossible based on a single anti-Stokes
frequency.
[0011] Spectroscopy imaging systems have also been developed in
which a broadband pulse is dispersed onto a multi-channel detector
(photodiode-array or CCD) after passing through the focus, such
that all spectral components can be individually detected. For
example, synchronized broadband and narrowband pulse trains may be
provided from mode-locked lasers. The combined pulse trains are
provided to a laser-scanning microscope, and the nonlinear sample
interaction occurs in the focus of the laser-scanning microscope.
Output radiation is then provided to a dispersion device such as a
grating or prism and then onto a multi-channel detector such as a
photodiode array of a CCD after passing through the focus. Because
of the use a spectrometer, images can only be achieved by slow
stage scanning or low-throughput de-scanned detectors.
[0012] Such an approach is also difficult to unify with the high
sensitivity detection schemes that require processing electronics
such as a lock-in amplifier because every spectral component would
need its own electronics. Furthermore spectroscopy is difficult to
combine with laser-scanning microscopy because after passing
through the sample the beam can move on the spectrometer and thus
hinder the spectrum acquisition.
[0013] There is a need, therefore, for a microscopy imaging system
that provides improved sensitivity and specificity. There is a
further need for a microscopy imaging system that probes multiple
Raman vibrations simultaneously to extract a spectral fingerprint
that is free from spectral interference from other atomic bonds
within a sample.
SUMMARY
[0014] In accordance with an embodiment, the invention provides a
microscopy imaging system that includes a light source system, a
spectral shaper, a modulator system, an optics system, an optical
detector and a processor. The light source system is for providing
a first train of pulses including a first broadband range of
frequency components, and a second train of pulses including a
second optical frequency such that a set of differences between the
first broadband range of frequency components and the second
optical frequency is resonant with a set of vibrational frequencies
of a sample in the focal volume. The second train of pulses is
synchronized with the first train of pulses. The spectral shaper is
for spectrally modifying an optical property of at least some
frequency components of the broadband range of frequency components
such that the broadband range of frequency components is shaped
producing a shaped first train of pulses to specifically probe a
spectral feature of interest from a sample, and to reduce
information from features that are not of interest from the sample.
The modulator system is for modulating a property of at least one
of the shaped first train of pulses and the second train of pulses
at a modulation frequency to provide a modulated train of pulses.
The optics system is for directing and focusing the shaped first
train of pulses and the second train of pulses as modulated toward
a common focal volume. The optical detector is for detecting an
integrated intensity of substantially all optical frequency
components of a train of pulses of interest transmitted or
reflected through the common focal volume. The processor is for
detecting a modulation at the modulation frequency of the
integrated intensity of substantially all of the optical frequency
components of the train of pulses of interest due to the non-linear
interaction of the shaped first train of pulses with the second
train of pulses as modulated in the common focal volume, and for
providing an output signal for a pixel of an image for the
microscopy imaging system.
[0015] In accordance with another embodiment, the system provides a
method of performing microscopy imaging using frequency modulation.
The method includes the steeps of providing a first train of pulses
at including a first broadband range of optical frequency
components; providing a second train of pulses including a second
optical frequency such that a set of differences between the first
broadband range of frequency components and the second optical
frequency is resonant with a set of vibrational frequencies of a
sample in the focal volume, wherein the second train of pulses is
synchronized with the first train of pulses; and spectrally
modifying an optical property of at least some frequency components
of the first broadband range of frequency components to provide a
shaped first train of pulses that is shaped to specifically probe a
spectral feature of interest from a sample, and to reduce
information from features that are not of interest from the sample.
The method further includes the steps of modulating an optical
property of one of the shaped first train of pulses and the second
train of pulses at a modulation frequency to provide a modulated
train of pulses and providing the other of the shaped first train
of pulses and the second train of pulses as a non-modulated train
of pulses; directing and focusing the modulated train of pulses and
the non-modulated train of pulses toward a common focal volume;
detecting an integrated intensity of substantially all optical
frequency components of the other of the modulated train of pulses
and the non-modulated train of pulses transmitted or reflected
through the common focal volume by blocking the modulated train of
pulses; detecting a modulation at the modulation frequency of the
integrated intensity of substantially all of the optical frequency
components of the non-modulated train of pulses due to the
non-linear interaction of the modulated train of pulses with the
non-modulated train of pulses in the common focal volume; and
providing the detected modulation as the signal for a pixel of an
image for a microscopy imaging system.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] The following description may be further understood with
reference to the accompanying drawings in which:
[0017] FIG. 1 shows an illustrative graphical representation of a
Raman spectrum of the bioactive molecule adenosine
triphosphate;
[0018] FIG. 2 shows an illustrative diagrammatic view of the
molecule adenosine triphosphate;
[0019] FIG. 3 shows an illustrative diagrammatic view of a
multiplex excitation microscopy system in accordance with an
embodiment of the invention;
[0020] FIG. 4 shows an illustrative diagrammatic view of the
generation of an excitation mask in accordance with an embodiment
of the invention;
[0021] FIGS. 5A-5C show illustrative diagrammatic views of response
signals from two components of a sample that may be interrogated in
accordance with an embodiment of the invention;
[0022] FIG. 6 shows illustrative graphical representations of Raman
shift verses intensity and magnitude for toluene and
cyclohexane;
[0023] FIG. 7 shows an illustrative graphical representation of
Raman shift verses stimulated Raman signal in a system in
accordance with an embodiment of the invention;
[0024] FIG. 8 shows an illustrative graphical representation of
Raman shift verses mask selectivity in a system in accordance with
an embodiment of the invention;
[0025] FIGS. 9A and 9B show illustrative diagrammatic
representations of input and output spectra respectively for a
stimulated Raman spectroscopy system in accordance with an
embodiment of the invention;
[0026] FIGS. 10A and 10B show illustrative diagrammatic
representations of input and output pulse trains respectively for a
stimulated Raman spectroscopy system in accordance with an
embodiment of the invention;
[0027] FIG. 11 shows an illustrative diagrammatic view of a
microscopy imaging system in accordance with an embodiment of the
invention;
[0028] FIG. 12 shows an illustrative diagrammatic view of signal
from a sample being reflected off of material within the sample
volume in a system in accordance with an embodiment of the
invention;
[0029] FIG. 13 shows an illustrative diagrammatic view of a portion
of a stimulated Raman spectroscopy system in accordance with
another embodiment of the invention employing epi-detection;
[0030] FIG. 14 shows an illustrative diagrammatic view of
vibrational states of a narrow-band stimulated Raman spectroscopy
system;
[0031] FIG. 15 show illustrative diagrammatic views of vibrational
states in a multiplexed stimulated Raman spectroscopy system in
accordance with an embodiment of the invention;
[0032] FIG. 16 shows an illustrative diagrammatic view of a
microscopy imaging system in accordance with a further embodiment
of the invention employing phase modulation;
[0033] FIG. 17 shows an illustrative diagrammatic view of the
generation of an excitation mask in accordance with a further
embodiment of the invention;
[0034] FIGS. 18A-18G show illustrative graphical data and images
associated with spectral SRS imaging obtained using a system in
accordance with an embodiment of the invention;
[0035] FIGS. 19A-19H show illustrative graphical data and images in
connection with lipid storage in Caenorhabditis Elegans obtained
using a system in accordance with an embodiment of the invention;
and
[0036] FIGS. 20A-20L show illustrative graphical data and images in
connection with label-free microscopy of absorbing samples obtained
using a system in accordance with an embodiment of the
invention.
[0037] The drawings are shown for illustrative purposes only.
DETAILED DESCRIPTION
[0038] In accordance with certain embodiments, the invention
provides excitation illumination that is spectrally and temporally
designed to probe specific species, and in further embodiments the
excitation illumination probes multiple chemical species
simultaneously. In accordance with some embodiments, the excitation
illumination systems may be employed with stimulated Raman
scattering microscopy systems.
[0039] The present invention, involves, in part, performing
excitation spectroscopy instead of detection spectroscopy for
Stimulated Raman Scattering (SRS) and Coherent Anti-Stokes Raman
Scattering (CARS) microscopy, as well as two-color two-photon
absorption and photothermal imaging, in order to overcome the above
cited shortcomings of prior art systems. Instead of providing a
complete broadband spectrum as the broadband excitation beam only
selected frequency components of one excitation mask may be
detected with a single detector, and the SRS may be extracted with
a high-frequency detection scheme. Spectral shaping has the
advantage that subsequent excitation masks may be applied without
having to change the laser. Different masks may be applied after
each imaging frame or on a pixel-by-pixel basis.
[0040] FIG. 3 shows a possible setup of an SRG microscope in which
excitation spectroscopy may be performed by pulse-shaping the
broadband beam. The system 14 includes a picoseconds (narrowband)
optical parametric oscillator 16, a synchronization unit 18, a
femtosecond (broadband) Ti:SA laser 20, and an amplitude modulator
22. The pump-beam is modulated at frequency f (>1 MHz) with the
amplitude modulator 22, and the pump and Stokes beams are provided
to a laser-scanning microscope 24. The pump and Stokes beams that
are transmitted or reflected through the focal volume are filtered
by the optical filter 26 to block the modulated pump beam and the
filtered Stokes beam 30 is detected by a photodetector such as a
photodiode 28. In various embodiments, the first train of pulses
may be femtosecond pulses and the second train of pulses may be
picosecond pulses or femtosecond pulses.
[0041] The modulation of the detected intensity of the Stokes beam
due to the nonlinear interaction with the sample is extracted with
an electronic processing unit such as a lock-in amplifier.
Excitation spectroscopy is performed by shaping the broadband pulse
by an amplitude or polarization pulse shaper 31 that consists of a
dispersive element 32 that disperses the individual frequency
components of the broadband beam onto the different elements of a
multiplex amplitude or polarization shaper such as a spatial light
modulator 36. Such a device can work in reflection mode (as shown)
or in transmission mode. Typically, a lens 34 is positioned in a
way to refocus the reflected beam such that an un-chirped,
spectrally homogenous beam is provided to the spatial light
modulator 36. In various embodiments, the settings on the pulse
shaper 31 may also be changed or modulated during imaging to
provide either a modulation of the pulse train or to provide
different sets of pulse shapes for probing multiple species within
a sample.
[0042] In line with the high-frequency modulation scheme presented
above and necessary for high-sensitivity SRS detection, amplitude
modulation is performed with an additional electro-optical or
acoustic-optical modulator, for example in the set-up shown in FIG.
3. Alternatively, it is possible that pulse-shaping of the
broadband beam and amplitude modulation for high-frequency
detection may be performed by the same device such as an
acousto-optic tunable filter (AOTF). Pump and Stokes beam may be
combined with a dichroic beam-combiner. If the pump-beam is
modulated for stimulated Raman gain (SRG) as discussed further
below, all frequency components of the Stokes beam are collected
with a single photodiode as described above and the SRG for a
particular excitation pattern is extracted with electronic
processing systems such as a lock-in detector to provide the pixel
of an image.
[0043] FIG. 4 shows how such an excitation mask may be generated
from a broadband laser spectra 40 and a pulse shaper 42 that
consists of a dispersive element (grating or prism) and a multiplex
amplitude shaper (such as a spatial light modulator SLM or printed
transmission/reflection mask) that can individually control the
intensity of every frequency components of the broadband excitation
pulse to provide a shaped train of pulses as shown at 44.
[0044] Such an approach can improve specificity by implementation
of a background subtraction scheme for interfering species. Instead
of illuminating the sample with one excitation mask, the signal for
two masks is measured. A first mask 1 contains mainly the frequency
components of the target molecule and a second mask 2 contains
mainly the frequency components of interfering species. Because of
the spectral interference of the target molecule with interfering
or other species in certain applications, the signal for mask/can
never be chosen to only contain contributions from the target
species but will always excite signal from the interfering or other
species. It is however, always possible to design the two
excitation masks in a way, that the difference between the
intensities for the two masks is independent of the concentration
of the interfering or other species. In accordance with further
embodiments, the two more species may be probed separately wherein
for each probing information from the non-probed species is
reduced.
[0045] The difference between the signal from mask 1 and mask 2 may
either be taken on a pixel by pixel basis or an image with mask 1
and mask 2 may be taken first and the subtraction may be performed
in the post-processing. The idea of this multivariate optical
computation has been used for emission spectroscopy for spontaneous
Raman scattering as shown in the prior art, but not for excitation
spectroscopy in combination with high sensitivity detection in SRS
or CARS.
[0046] In accordance with further embodiments, pulse shaping may be
achieved using a multiplex electro-optic modulator, a multiplex
electro-acoustic modulator, an acousto-optic tunable filter, or a
Dazzler system as sold by Fastlite Societe a responsabilite limitee
of Saint-Aubin France. The broadband beam is not required to have
all frequency components within a range present in the beam, but
instead may be composed of a plurality of center frequency
components, as long as sufficient frequency components are present
in the broadband beam that may be shaped for probing a sample as
desired. The spectral range of the broadband beam may be, for
example, at least 15.0 nm, or at least 5.0 nm, or at least 1.0 nm,
or even at least 0.5 nm in certain embodiments.
[0047] FIGS. 5A to 5C show three fundamental scenarios of possible
spectral crosstalk between two species. In FIG. 5A the only strong
peak of the target molecule component A at .omega..sub.A as shown
at 50 overlaps with the spectrum of another species (component B in
the sample) as shown at 52 and 54 that has a second peak at
.omega..sub.B. In this situation the first excitation mask is
chosen such that it gives maximum signal for the peak at
.omega..sub.A, yielding the signal from component A and component
B. The second excitation mask is chosen in a way that it removes
the interfering signal from component B after subtracting the
signal from the first mask and the second mask.
[0048] The masks may be designed in such a way, that the signal
from the isolated peak of the interfering species is scaled in such
a way that after subtraction this only leaves the pure signal from
the target species, which is independent of the concentration of
component B. FIG. 5B shows at 56 and 58 the case of a spectrally
shifted target peak due to the chemical environment. In this
situation the two excitation masks can be chosen in a way that the
original peak from the un-shifted species does not contribute to
the signal after subtraction (e.g., signal from the left half of
the peak minus signal from the right half of the peak). In FIG. 5C
the spectra from the target species as shown at 60 sits on top of a
spectrally flat background as shown at 62. Such a background may
arise from other nonlinear processes as Kerr-lensing and two-color
two-photon absorption or a strongly broadened vibrational resonance
in SRS and from the non-resonant background in CARS. The two masks
can be chosen to maximize the signal from the peak and subtract the
flat background.
[0049] FIG. 6 shows how the scenarios illustrated in FIGS. 5A-5C
may be applied to more complex spectra and more than one
interfering species. In particular, FIG. 6 shows at 64 the spectra
for toluene, and shows at 66 the positive mask to probe for
toluene, while 68 shows a negative mask to exclude an interfering
species. FIG. 6 also shows at 70 the spectra for cyclohexane, and
shows at 72 the positive mask to probe for cyclohexane, while 74
shows a negative mask to exclude an interfering species. This
approach has been applied to emission spectroscopy and is known as
`multivariate optical calculation`. It is based on treating spectra
as N dimensional vectors, where N is the number individual
frequency components of the excitation pulse, and the applied masks
as co-vectors.
[0050] It is also desirable to comprise a concrete implementation
for fast switching between the two excitation masks for a
pixel-by-pixel subtraction. Because laser noise occurs primarily at
frequencies<1 MHz, the difference between the excitation masks
needs to be taken at fast rates to achieve maximum sensitivity for
the target molecule. The approach is based on polarization-shaping
the broadband pulse (s-polarization corresponds to mask 1 and
p-polarization corresponds do mask 2) and switching between the two
polarization states, i.e., excitation masks, with an electro-optic
modulator (Pockel cell). A similar approach may be used in an
implementation of frequency modulation CARS.
[0051] A more complex scenario is shown in FIG. 7. Molecule A
(shown at 75) is the analyte of interest, while elements B (shown
at 76) and C (shown at 76) are the interfering or other species.
Note that the corresponding known Raman spectra
.sigma.(.DELTA..omega.) of A, B, and C are partially overlapping
with each other as shown.
[0052] The objective is to design positive excitation spectral
shapes. In a mixture of A, B and C with unknown concentrations c of
each one, two positive excitation spectral shapes
I.sub.+(.DELTA..omega.) and I.sub.-(.DELTA..omega.) (i.e. masks)
may be designed such that the difference signal .DELTA.S from these
two excitation masks can selectively predict the concentration of
molecule A without getting interference from molecules B and C.
[0053] For a given excitation spectral shape I(.DELTA..omega.), the
obtained absorption signal S will be described by the following
S.varies..intg.(.DELTA..omega.)[c.sub.A.epsilon..sub.A(.DELTA..omega.)+c-
.sub.B.epsilon..sub.B(.DELTA..omega.)+c.sub.CE.sub.C(.DELTA..omega.)]d.DEL-
TA..omega.
[0054] For two excitation spectral shapes I.sub.+(.DELTA..omega.)
and I.sub.-(.DELTA..omega.), the difference signal .DELTA.S will
be
.DELTA.S.ident.S.sub.+-S.sub.-.varies..intg.[I.sub.+(.DELTA..omega.)-I.s-
ub.-(.DELTA..omega.)][c.sub.A.epsilon..sub.A(.DELTA..omega.)+c.sub.B.epsil-
on..sub.B(.DELTA..omega.)+c.sub.C.epsilon..sub.c(.DELTA..omega.)]d.DELTA..-
omega.
It is mathematically possible that we can design positive
I.sub.+(.DELTA..omega.) and I.sub.-(.DELTA..omega.) functions such
as their difference function satisfy the following orthogonal
relations with the Raman spectra of all the interferent
species:
.intg.[I.sub.+(.DELTA..omega.)-I.sub.-(.DELTA..omega.)].epsilon..sub.B(.-
DELTA..omega.)d.DELTA..omega.=0 and
.intg.[I.sub.+(.DELTA..omega.)-I.sub.-(.DELTA..omega.)].epsilon..sub.C(.D-
ELTA..omega.)d.DELTA..omega.=0
Note that the Raman shift-dependent
I.sub.+(.DELTA..omega.)-I.sub.+(.DELTA..omega.) has both positive
and negative values. As a result, we can simplify the quantity
.intg.[I.sub.+(.DELTA..omega.)-I.sub.-(.DELTA..omega.)][c.sub.A.epsilon.-
.sub.A(.DELTA..omega.)+c.sub.B.epsilon..sub.B(.DELTA..omega.)+c.sub.C.epsi-
lon..sub.C(.DELTA..omega.)]d.DELTA..omega.=.intg.[I.sub.+(.DELTA..omega.)--
I.sub.-(.DELTA..omega.)]c.sub.A.epsilon..sub.A(.DELTA..omega.)d.DELTA..ome-
ga.
[0055] The difference signal therefore, is only proportional to the
concentration of molecule of interest:
.DELTA.S.ident.S.sub.+-S.sub.-.varies.c.sub.A.intg.[I.sub.+(.DELTA..omeg-
a.)-I.sub.-(.DELTA..omega.)].epsilon..sub.A(.DELTA..omega.)d.DELTA..omega.
[0056] FIG. 8 shows a design for excitation spectral shapes using
masks I.sub.+(.DELTA..omega.) (shown at 78) and
I.sub.-(.DELTA..omega.) (shown at 79). The mask
I.sub.+(.DELTA..omega.) 78 is designed to enhance imaging of the
element of interest, while the mask I.sub.-(.DELTA..omega.) 79 is
designed to reduce the imaging of interfering elements.
[0057] Because changing different components of excitation spectrum
by pulse-shaping does not require the laser to change, fast
switching speeds are possible allowing for spectra-temporal shaping
of the excitation beams to encode the signal from different
chemical species in time (or modulation phase and frequency)
instead of optical frequency such that it can be detected with a
single detector. This allows implementation of: 1) real-time
detection of pure signal from target species free from the
background signal of interfering species by the subtraction scheme
of the two-masks as described above, and 2) simultaneous multicolor
imaging with a single detector.
[0058] It is desirable to comprise a concrete implementation for
fast switching between multiple excitation, masks, because laser
noise occurs primarily at frequencies<1 MHz and different
excitation masks need to be probed faster than the laser can
change. The sample may also change in between frames (e.g., move),
making a frame-by-frame acquisition of different mask
impossible.
[0059] Technically there are many multiple technologies to achieve
such fast spectral modulations, such as: 1) spectral modulation
with a single device such as an electro-optic or acousto-optic
modulator that allows the independent modulation of individual
spectral component of the broadband excitation beam, and 2) a
combination of a polarization pulse shaper, polarization modulator
and polarization analyzer.
[0060] Systems of the invention may be employed with stimulated
Raman scattering (SRS) microscopy systems as follows. Stimulated
Raman scattering allows the detection of the vibrational signal
with higher signal levels than spontaneous Raman scattering due to
stimulated excitation of molecular vibrations and without exciting
the non-resonant background signal of CARS microscopy. Spontaneous
Raman spectra are thus preserved and the signal strength scales
linearly with the concentration allowing for straight forward
quantification. Forward- and reverse (epi)-detection is possible,
as well as SRS endoscopy.
[0061] In a narrowband SRS microscopy, pump and Stokes-beam are
used to excite the sample, just as in CARs microscopy. Instead of
detecting the newly emitted light at the anti-Stokes frequency,
intensity gain (stimulated Raman gain) at the pump frequency or
intensity loss (stimulated Raman loss) at the Stokes frequency are
detected. As the gain and loss are relatively small a
high-frequency detection scheme is often required, in which the SRS
signal is modulated at a known frequency that is higher than the
laser noise and is extracted with an electronic detector such as a
lock-in amplifier to provide the intensity of a pixel. The
modulation may be frequency modulation, phase modulation or
amplitude modulation.
[0062] In this narrowband approach to coherent Raman imaging (CARS
and SRS microscopy) only a single molecular vibration can be imaged
at a time. Thus only single color images can be produced (compared
to imaging multiple species simultaneously) and detection is
limited to chemical species that have an isolated vibration that
does spectrally not interfere with other compounds in the sample.
The present invention provides (in certain embodiments) methods and
systems to allow coherent Raman imaging based on multiple Raman
lines simultaneously.
[0063] For example, fast, label-free imaging of biological samples
based on vibrational signatures of the target molecules is possible
with SRS. High sensitivity (measurement of intensity changes
.DELTA.I/I<10.sup.-7) even with noisy lasers may be achieved by
implementation of high-frequency (>1 MHz) signal modulation and
phase sensitive detection with a lock-in amplifier, because laser
noise primarily occurs at lower frequencies. For SRS imaging a
single vibrational frequency is selected by tuning the frequency
difference of the two-excitation lasers. The spatial distribution
of the target molecule in the sample can be probed with a
laser-scanning microscope by raster-scanning the laser-focus
through the sample. Vibrational excitation spectra from a single
point in the sample can be obtained by automated tuning of the
excitation laser frequency. Excitation pulsed lasers (.about.5 ps)
are useful that provide high peak intensities (favoring the
nonlinear optical interaction at the low average intensity required
for biomedical imaging) and provide high enough spectral resolution
(.about.3 cm.sup.-1) to time into selected vibrational bands even
if the `integrated intensity of substantially all the optical
frequency components of the laser is collected.
[0064] In particular, a pump beam and a Stokes beam in a sample
volume enhance a spontaneous Raman radiation signal. The center
frequency of the Stokes beam and the center frequency of the pump
beam are separated by an input spectra difference .OMEGA. as shown
at 80 and 82 in FIG. 9A. As shown in FIG. 9B, SRS leads to an
intensity increase in the Stokes beam (stimulated Raman gain or
SRG) and an intensity decrease in the pump beam (stimulated Raman
loss or SRL) as shown at 84 and 86. Also shown (not to scale) is
the CARS signal 88 that is generated at the anti-Stokes frequency
.omega..sub.AS.
[0065] FIGS. 10A and 10B illustrate the SRL detection scheme. The
pump beam is provided as an input pulse train 90, and the Stokes
beam is provided as an input pulse train 92 that is modulated at
high frequency f (MHz). The output pulse train (shown at 94)
includes a resulting amplitude modulation at the high frequency
(MHz) due to stimulated Raman loss (SRL) that can only occur if
both beams are present. This modulation of the originally
non-modulated beam at the same frequency of the modulation f may
then be detected by detection electronics and separate it from the
laser noise that occurs at other frequencies. Stimulated Raman gain
(SRG) of the Stokes-beam can be probed by modulating the pump beam
and detecting the Stokes beam.
[0066] An SRL microscope may be provided with either or both
forward and epi (reverse) detection. The Stokes beam may be
modulated by an electro-optic (or acoustic-optic) modulator and
then combined with the pump beam by a beam splitter/combiner. The
collinear pump and Stokes beam are then positioned by an x-y
scanner system, and directed toward a sample. The transmitted or
reflected pump beam is filtered by a filter, and detected by a
photodiode (PD). For epi detection, the back-scattered beams are
collected by the excitation objective lens (OL) and separated from
the excitation beams by a combination of a quarter wave plate
(.lamda./4) and polarizing beam splitter (PBS). For forward
detection, the forward-scattered beams are collected by a
condenser. The SRL is measured by a lock-in amplifier to provide a
pixel of the image. Three dimensional (3D) images are obtained by
raster-scanning the laser focus across the sample by the scanner
system and microspectroscopy can be performed by automated tuning
of the pump wavelength.
[0067] In accordance with various embodiments, the microscopy
system may be provided using a variety of sources and a variety of
modulation techniques. FIG. 11, for example, shows a microscopy
imaging system 100 in accordance with an embodiment of the
invention that includes a dual frequency laser source 102 and an
optical parametric oscillator 104. The dual frequency laser source
102 provides a broadband train of laser pulses 106 at a center
frequency (e.g., including a Stokes frequency .omega..sub.1 of, for
example, 1064 nm), and a train of laser pulses 108 at a more narrow
band of frequencies having a center frequency (e.g., 532 nm) to the
optical parametric oscillator 104. The optical parametric
oscillator may be, for example, as disclosed in U.S. Pat. No.
7,616,304, the disclosure of which is hereby incorporated by
reference in its entirety. The output of the optical parametric
oscillator provides a train of laser pulses 110 at a center
frequency .omega..sub.2 (e.g., a pump frequency) that is selected
such that a difference between .omega..sub.1 and .omega..sub.2
(e.g., .omega..sub.p-.omega..sub.S) is resonant with a vibrational
frequency of a sample 112 in a focal volume.
[0068] Each pulse of the train of laser pulses 106 is then
spectrally shaped by a shaping assembly 114 that includes a
dispersive element 116, a lens 118 and a spatial light modulator
120. The dispersive element 116 spectrally disperses each broadband
pulse, and the spatial light modulator 120 then modulates different
frequency components of the spectrally disperse broadband pulse to
provide a train of shaped pulses 122.
[0069] The train of shaped laser pulses 122 is then modulated by a
modulator 124, and is then phase adjusted at a translation stage
126 (that is adjustable as indicated at A) to ensure that the
resulting train of modulated shaped laser pulses 128 and the train
of laser pulses 110 at the center pump frequency are temporally
overlapped. The two trains of laser pulses 128 and 110 are combined
at a combiner 130 such that they are collinear and spatially
overlapped as well.
[0070] The combined trains of laser pulses 128 and 110 are directed
via a scan-head 132 (that scans in mutually orthogonal x and y
directions), into a microscope 134 that includes optics 136 that
direct and focus the combined trains of laser pulses 128 and 110
into the focal volume, e.g., via a mirror 138. The illumination
from the focal volume is directed by a condenser 140 onto an
optical detector 142 (e.g., a photodiode), and the modulated shaped
beam (e.g., the Stokes beam) is blocked by an optical filter 144,
such that the optical detector 142 measures the intensity of the
other beam .omega..sub.1 (e.g., the pump beam) only.
[0071] The train of shaped laser pulses 122 is modulated at
modulation frequency f (e.g., at least about 100 kHz), by a
modulation system that includes, for example, the modulator 124, a
controller 146 and a modulation source 148. The modulation source
provides a common modulation control signal 150 to the controller
146 as well as to a signal processor 152. The integrated intensity
of substantially all frequency components of the first pulse train
154 from the optical detector 142 is provided to the signal
processor 152, and the intensity modulation due to the non-linear
interaction of the train of laser pulses 128 with the train of
laser pulses 110 in the focal volume is detected at the modulation
frequency f to provide a pixel of an image to a microscopy control
computer 156. The microscopy control computer 156 is employed as an
imaging system, and further provides user control of the scanhead
132 as shown at 158.
[0072] In accordance with an embodiment, the modulation system may
provide amplitude modulation of the shaped pulses to provide the
modulated shaped pulse train 128 such that only alternating pulses
of the shaped pulse train 122 are coincident with the pulses of the
.omega..sub.1 pulse train 110. Such amplitude modulation of the
shaped beam may be achieved using a Pockel cell and polarization
analyzer as the modulator 124, and a Pockel cell driver as the
controller 146. In accordance with another embodiment, the
modulation rate is half the repetition rate of the laser such that
every other pulse of the original .omega..sub.2 pulse train is
reduced in amplitude to provide that stimulated Raman scattering
does not substantially occur in the focal volume with the pulses
having the reduced amplitude. If the modulation rate is of the same
order of the repetition rate of the laser, countdown electronics
can be utilized to guarantee the synchronization (phase) between
the modulation and the pulse train. Lower modulation rates are also
possible, as long as the modulation frequency is faster that the
laser noise. In further embodiments, the contrast pulses may have
an amplitude that is substantially zero by switching off the pulses
at the modulation frequency, for example using an electro-optic
modulator or an acousto-optic modulator.
[0073] Amplitude modulation of the pump or Stokes pulse trains may
therefore be achieved, and the increase of the Stokes pulse train
or decrease of the pump pulse train may be measured. By modulating
the pump train of pulses and then detecting the Stokes train of
pulses from the focal volume, stimulated Raman gain (SRG) may be
determined by the processing system. In further embodiments, the
Stokes beam may be modulated, the pump beam may be detected from
the focal volume, and stimulated Raman loss (SRL) may be determined
by the processing system. In still further embodiments, the phase
of one of both the shaped beam 122 and the non-shaped beam 110 may
be phase modulated or frequency modulated as long as the modulation
is done at the modulation frequency such that the detection system
is able to extract the signal of interest. In still further
embodiments, both the pump and Stokes beams may be modulation by a
modulation system.
[0074] Systems of various embodiments of the invention, therefore,
provide that stimulated Raman scattering microscopy may be achieved
using a modulation of one of the pump or Stokes beams as a contrast
mechanism. Stimulated Raman scattering microscopy bears most of the
advantages of the existing methods. In particular, 1) it is a
optically stimulated process which significantly enhances the
molecular vibrational transition efficiency compared to
conventional Raman microscopy which relies on spontaneous
scattering; 2) it is a nonlinear process in which the signal is
only generated at the microscopy objective focus, rendering a
three-dimensional sectioning ability; 3) it only probes the
vibrational resonance, and it is free from interference with the
non-resonant background, unlike in the CARS microscopy where
non-resonant background is always present; 4) the signal always
scales linearly with the solute concentration, allowing ready
analytical quantification; 5) the signal can be free from sample
auto-fluorescence; 6) the phase matching condition is always
satisfied for any relative orientations of the beams, unlike in the
CARS microscopy; 7) visible and near-IR beams are used resulting in
a higher penetration depth and spatial resolution than IR
absorption microscopy; and 8) the detection of stimulated Raman
gain or loss is also unaffected by ambient light, which permits
such systems to be used in open environments.
[0075] The process may be viewed as a two photon process for
excitation of a vibrational transition. The joint action of one
photon annihilated from the pump beam and one photon created to the
Stokes beam promotes the creation of the molecular vibrational
phonon. The energy of the pump photon is precisely converted to the
sum of the energy of the Stokes photon and the molecular
vibrational phonon. As in any two photon optical process, the
transition rate is proportional to the product of the pump beam
intensity and the Stokes beam intensity. It is obvious that a
molecular vibrational level is necessary for this process to
happen, as required by the energy conservation. There is,
therefore, no contribution from non-resonant background would be
present. This represents a significant advantage over CARS
microscopy which is severely limited by non-resonant background
which not only distorts the spectrum but also carries unwanted
laser noise.
[0076] The process may also be treated as a stimulated version of
the spontaneous Raman scattering. In spontaneous Raman scattering,
the Stokes photon mode is empty in the initial state and the vacuum
field serves as the stimulated Stokes beam. That is why the
efficiency is extremely low. The transition rate is only
proportional to the pump beam intensity. In stimulated Raman
scattering however, the Stokes photon mode has a large number of
pre-occupied photons due to the presence of a strong laser beam,
and the scattering process becomes stimulated in analogy to the
stimulated emission. As a result, the transition rate is
proportional not only to the pump beam intensity as in spontaneous
Raman scattering, but also to the number of pre-occupied photons in
Stokes photon mode which is again proportional to the Stokes beam
intensity.
[0077] The process may also be accounted for as a heterodyne
interference between the pump beam (or the Stokes beam) and a
corresponding third-order nonlinear induced radiation at the same
optical frequency as the pump beam (or the Stokes beam). These two
third-order nonlinear induced polarizations for stimulated Raman
gain and loss are different from each other, and are also distinct
from the one responsible for CARS generation. If there are no
additional electronic resonances involved, however, their absolute
sizes are all the same.
[0078] For stimulated Raman loss of the pump beam, this third-order
nonlinear induced polarization radiates at the pump beam frequency.
The intensity dependence of this nonlinear radiation scales
linearly with pump beam and quadratically with Stokes beam. Its
final phase is 180 degree lag behind that of the input pump beam at
the far field detector. Therefore, the interference between this
nonlinear radiation and input pump beam results in an attenuation
of the pump beam itself. And the intensity dependence of the
interference term scales linearly with both the pump beam and
Stokes beam.
[0079] For stimulated Raman gain for Stokes beam, a different
third-order nonlinear induced polarization radiates at the Stokes
beam frequency. The intensity dependence of this nonlinear
radiation scales quadratically with pump beam and linearly with
Stokes beam. Its final phase is the same as that of the input
Stokes beam at the far field detector. Therefore, the interference
between this nonlinear radiation and input Stokes beam results in
an increase of the Stokes beam itself. The intensity dependence of
the interference term again scales linearly with both the pump beam
and Stokes beam.
[0080] Although the use of amplitude modulation has the highest
modulation depth, this approach may introduce a linear background
due to a modulation of the temperature or refractive index of the
sample due to the intensity modulation on the sample. In accordance
with another embodiment, the modulation system may provide
polarization modulation, and may include a polarization device as
the modulator, and a polarization controller as the controller.
Every other pulse of the .omega..sub.2 pulse train has a
polarization that is different than that of the other preceding
pulse. Each of the .omega..sub.t pulses of the pulse train is
coincident with a .omega..sub.1 pulse of the .omega..sub.1 pulse
train. Different modulation rates other than half of the repetition
rate of the laser (in which every other pulse is different) can
also be applied.
[0081] Polarization modulation also provides that stimulated Raman
scattering does not substantially occur in the focal volume with
the pulses having the altered unparallel polarization. In certain
embodiments, the modulator includes a polarization filter to remove
one of the sets of pulses as a further contrast mechanism. The
polarization of the pulses may therefore, be modulated with respect
to each other. In other embodiments, the detector itself may
distinguish between the modulated pulses. In particular, when pump
and Stokes pulse trains are perpendicular to each other, a
different tensor element of the nonlinear susceptibility is probed
compared to the case where pump and Stokes field are parallel.
Different tensor elements have significantly different magnitudes.
This converts the polarization modulation of the excitation beams
into amplitude modulations of the gain/loss signal which can then
be detected with the lock-in amplifier. Polarization modulation can
be implemented with a Pockel cell. This approach has the advantage
that it does not introduce a temperature modulation of the
sample.
[0082] In accordance with other embodiments, one of the pulse
trains may be modulated by time-shifting (or phase). For example,
one pulse train may include alternating pulses that coincide with a
.omega..sub.1 pulse, while the remaining pulses are time shifted
such that they do not coincide with a .omega..sub.1 pulse.
Modulation of one or both of the pump and Stokes beams may also be
achieved by frequency modulation as disclosed for CARS microscopy,
for example, in U.S. Pat. No. 7,352,458, the disclosure of which is
hereby incorporated by reference in its entirety. In a frequency
modulation system, the frequency of one or both of the pump and
Stokes beams is alternately modulated at a modulation frequency
such that a difference frequency between the pump and Stokes beams
(e.g., .omega..sub.p-.omega..sub.S) is tuned in and out of a
vibrational frequency of the sample. The detector then detects the
gain/loss that is generated through non-linear interaction of
.omega..sub.p and .omega..sub.S and the sample responsive to the
modulation frequency. An output signal may be passed through a
lock-in amplifier such that only changes at the time scale of the
modulation period are provided in the final output. In accordance
with further embodiments, other modulation schemes may be employed
such as time-delay modulation, spatial beam mode modulation, etc.,
which will each introduce a modulation of a generated signal.
[0083] For example, in accordance with further embodiments, systems
of the present invention may employ a dual frequency laser source,
a first optical parametric oscillator, as well as an additional
optical parametric oscillator that splits the power of the dual
frequency laser source. The dual frequency laser source provides a
first train of laser broadband pulses (including a pump frequency
.omega..sub.1) and a second train of laser pulses at a center
frequency to the optical parametric oscillator and to the optical
parametric oscillator. The first train of laser pulses are shaped
as discussed above with reference to FIG. 11. The output of the
optical parametric oscillator provides a third train of laser
pulses at a center Stokes frequency .omega..sub.2 that is selected
such that a difference between .omega..sub.1 and .omega..sub.2
(e.g., .omega..sub.p.omega..sub.S) is resonant with a vibrational
frequency of a sample (not shown) in a focal volume. The output of
the optical parametric oscillator provides a fourth train of laser
pulses at a center frequency .omega..sub.2' that is selected such
that a difference between .omega..sub.1 and .omega..sub.2 (e.g.,
.omega..sub.p-.omega..sub.S') is not resonant with a vibrational
frequency of the sample in the focal volume.
[0084] The .omega..sub.2' pulses are passed through a half wave
plate and combined with the .omega..sub.2 pulses, which are passed
through a different half wave plate. The half wave plates ensure
that the pulse trains have different polarization such that one is
transmitted by the beam splitter and the other is reflected. At
this point, the combined pulse train includes both the
.omega..sub.2 and the .omega..sub.2' pulses, but with mutually
orthogonal polarization. The combined .omega..sub.2 and the
.omega..sub.2' pulses are passed through a modulator that,
responsive to a modulation signal that provides a modulation
frequency f from a modulation source. Based on the different
polarization the modulator together with a polarization analyzer
selects a different polarization at the modulation rate f, i.e., it
selects .omega..sub.t or .omega..sub.2' pulses. The result is that
a pulse train of alternating .omega..sub.2 and .omega..sub.2'
pulses is provided. The first shaped train of laser pulses and the
alternating train of laser pulses and are combined at a combiner
such that they are collinear and spatially overlapped as well, and
the combined pulse trains are directed toward a sample as discussed
above with reference to FIG. 11.
[0085] In accordance with further embodiments, the system may
include an electronically locked laser such as an electronically
locked mode-locked titanium sapphire laser in place of the optical
parametric oscillator. In still further embodiments, the system may
include a single optical parametric oscillator for providing both
the .omega..sub.2 and the .omega..sub.2' pulses, and the single
optical parametric oscillator may provide the alternating train of
laser pulses responsive to a modulation signal that is coupled to
the signal processor. In accordance with further embodiments, the
system may provide different spectral masks at different modulation
frequencies, as well as multiple lock-in detectors tuned to the
different modulation frequencies such that a plurality of species
may be probed at the same time.
[0086] In still further embodiments, the microscopy imaging system
may provide spectral-temporal excitation shaping in a CARS system.
With reference again to FIG. 11, is such a CARS system, the shaped
and modulated pump and/or Stokes trains of pulses 110, 128 are
still directed toward the sample 112 in the focal volume, but the
optical detector 142 and optical filter 144 are chosen such that
the anti-Stokes pulses are received at the detector 142. In
accordance with further embodiments, the optical detector 142 and
filter 144 are selected such that two-color two-photon absorption
is detected, or are chosen such that photo-thermal detection is
provided.
[0087] As shown in FIG. 12, during the non-linear interaction of
the modulated Stokes train of pulses (shown diagrammatically at
128) and the pump train of laser pulses (shown diagrammatically at
110) when focused through optics 136 toward a focal area 162, both
the pump and Stokes pulses are directed in a forward direction (as
show diagrammatically at 166). A detector that is positioned
forward of the sample will detect forward directed Stokes pulses
that pass through the sample.
[0088] As also shown in FIG. 12, during the non-linear interaction
of the shaped and modulated Stokes train of pulses 128 and the pump
train of laser pulses 110 when focused through optics 136 toward
the focal area generally shown at 162, some pump and Stokes pulses
are initially forward directed (as shown diagrammatically at 166)
but are then reflected by non-uniformities 168 within the sample
(as show diagrammatically at 170) back toward the optics 136. In
accordance with other embodiments therefore, a detector may also be
positioned in the reverse direction with respect to the incoming
pump and Stokes pulse trains that are directed into the focal
volume. In such as reverse direction detection system, the detector
will detect reflected pump pulses.
[0089] FIG. 13 shows a portion of system in accordance with a
further embodiment of the invention in which system components
having the same reference numerals as used in FIG. 11 (e.g., 110,
128, 112, 132, 136, 150, 152, 156, and 158) are the same as those
in FIG. 11. The remaining elements from FIG. 11 not shown in FIG.
13 are the same, and the system may provide amplitude modulation,
polarization modulation or frequency modulation as discussed
above.
[0090] The system of FIG. 13, however, employs an optical detector
142' that receives via a filter 144' an integrated intensity of the
optical frequency components of the train of Stokes pulses that are
reflected through the common focal volume. In particular, the
optics 136 directs and focuses the two trains of laser pulses into
a sample 112 at the focal volume, and illumination that is directed
in the epi-direction (by reflecting off other material in the
sample following Raman scattering) is directed back through the
optics 136 and beam splitter 138' onto the optical detector 142'
via filter 144'. The image signal 154 is provided to the signal
processing unit 152, which is in communication with the microscopy
control computer 156 as discussed above with reference to FIG.
11.
[0091] As the signal and excitation beams have the same optical
frequency, the system may provide that the beam splitter 138' is a
50/50 splitter that reflects 50% of an incident beam and transmits
50% of the incident beam through the beam splitter onto a heat
absorber 172. This would ideally provide that 25% of the Stokes
beam would be transmitted back into the detector 142'. In further
embodiments, the beam splitter 138' may be a 20/80 splitter that
reflects 20% of an incident beam and transmits 80% of the incident
beam through the beam splitter, resulting in 4% signal on the
detector 142'.
[0092] As with the embodiments discussed above, the system may
provide modulation at a modulation frequency f, such as amplitude
modulation, polarization modulation, phase modulation or frequency
modulation, and the processor 152 detects a modulation (amplitude
and/or phase) of the integrated intensity of substantially all of
the optical frequency components of the Stokes pulse train due to
the non-linear interaction of the Stokes pulse train with the pump
pulse train within the common focal volume.
[0093] The specificity of the SRS signal for a certain target
species of the presented single-band approach with narrowband
lasers is, however, limited, as different chemical bonds may have
the same vibrational frequencies. The full specificity for Raman
spectroscopy may be exploited only if the full vibrational spectrum
of all bonds of a compound (e.g., as shown at 10 in FIG. 1) are
probed rather than simply a single frequency.
[0094] In accordance with various embodiments of the invention
therefore, spectral masks may be used to provide improved imaging.
With reference for example, to the Raman spectrum shown at 10 in
FIG. 1, none of the individual peaks is isolated from those other
molecules, but the molecule's overall vibrational fingerprint,
however, is unique. Complex molecules have several Raman active
peaks, which combined result in a characteristic vibrational
signature of the molecules. Vibrational spectra can thus be used as
a label-free contrast mechanism for biomedical imaging.
[0095] If only a single Raman peak is used as a marker-band,
crosstalk between different compounds is possible. This can limit
the specificity of the methods in many cases. It is possible to
probe the a bigger portion Raman spectrum with SRS, by using at
least one broadband laser source as pump- and Stokes beam. FIG. 14
shows an energy state diagram for narrow-band SRS using a
narrow-band pump beam 180 and a narrow-band Stokes beam 182 wherein
multiple vibration states exist as shown at 184. Every spectral
component of the narrowband beam (frequency span is small than the
width of the Raman line) experiences a gain or loss due to SRS,
depending on whether the frequency difference of the center
frequencies corresponds to a vibrational frequency of the molecules
in the sample. The difference frequency is tuned into the resonance
frequency of one oscillation of the target molecule. The other
vibrational states are unaffected. As a result of the interaction
with the sample, the pump-beam experiences a loss (SRL) and the
Stokes beam a gain (SRG).
[0096] FIG. 15 shows two multiplex spectroscopy systems in which a
narrow-band pump beam 190 is used with a broadband Stokes beam 192
to probe the vibrational states shown at 194. The individual
components of broadband Stokes beam 192 and substantially all the
frequency components of the narrowband pump beam 190 experience SRG
or SRL respectively depending on whether the individual difference
frequencies corresponds to on of the molecular vibrational states
of the molecule 194.
[0097] In other embodiments as also shown in 15, a broadband pump
beam 196 may be used with a narrow-band Stokes beam 198 to probe
the vibrational states 200. The individual components of broadband
pump beam 196 and substantially all the frequency components of the
narrowband Stokes beam 198 experience SRL or SRG respectively
depending on whether the individual difference frequencies
corresponds to one of the molecular vibrational states of the
molecule 200. Broadband excitation is also possible by two
broadband pulse trains as pump and Stokes beams. The combination of
all spectral components of both broadband beams contribute to the
generation of the SRS signal.
[0098] As discussed with reference to FIG. 6 above, suppression of
spectral cross-talk can be achieved by subtracting the signal from
mask 2 (mainly containing the spectral components resonant with the
interfering molecules) from the signal from mask 1 (mainly
containing the spectral components resonant with the target
molecules). As laser noise scales with the absolute signal, i.e.,
with the signal from the target component and the interfering
species, the signal from the target molecules can easily be buried
in the laser noise of the interfering species, when its
concentration is much lower or the Raman scattering cross-section
is much weaker. For this reason the subtraction from mask/and mask
2 has to be accomplished at a MHz rate since laser noise occurs
mainly at lower frequencies. As such multivariate optical
computation applied to excitation spectroscopy in SRS microscopy is
equivalent to a complex frequency modulation scheme between two
arbitrarily shaped excitation spectra.
[0099] FIG. 16 shows a high-frequency modulation system in
accordance with another embodiment of the invention for
multivariate optical computation in an SRS microscope. FIG. 16
shows a setup for a SRG microscope system with pixel-by-pixel mask
subtraction. The system includes an illumination source system 210
that includes a picosecond (narrowband) optical parametric
oscillator 212, an electronic synchronization unit 214, and a
femtosecond (broadband) Ti:Sa laser 216. Excitation shaping is
performed on the broad band pulse using a polarization pulse shaper
218 (containing a dispersive element such as a grating 220, an
imaging lens 222 and a multiplex polarization shaper as a spatial
light modulator 224).
[0100] The system also includes an analyzer 226 that only passes
one polarization. A polarization modulator 228 (e.g., Pockel cell)
is positioned in front of the analyzer and switches between which
polarization is transmitted by the analyzer 226. As such it can
switch between different spectral components of the broadband pulse
depending whether the individual frequency components are set to be
in the one or the other polarization state by the polarization
pulse shaper. Electro-optical modulators such as Pockel cells allow
switching at rates>1 MHz as desired. The shaped broadband pulse
is overlapped with the narrowband Stokes pulse with a dichroic
beam-combiner and aligned into a beam-scanning microscope 230.
After passing or reflected through the focal volume of the focusing
optics, the modulated pump beam is blocked by a filter 232 and the
Stokes beam is detected with a photodetector such as a photodiode
234. The SRG on the originally non-modulated Stokes beam caused by
the nonlinear interaction due to just the target molecule in the
focus of the laser scanning microscope, can then be extracted with
processing electronics such as a lock-in amplifier 236, detecting
at the modulation rate of the electro-optic modulator. The lock-in
amplifier takes the difference between the two spectra
automatically. No additional modulation is necessary. A
three-dimensional image of the distribution of just the target
compound can then be acquired by scanning the focus through the
sample.
[0101] As also shown in FIG. 16, the imaging system also includes
an input device 240, a controller 242 that is coupled to a memory
storage unit 244 and to the pulse-shaper 218. The signal processor
236 is also coupled to an output display device 238. A user may
input at unit 240 an identification of an element to be analyzed,
and the controller 242 may then obtain from a storage device 244
(e.g., via direct connection or via a network) the spectral shaping
information associated with the element. The controller 242 then
directs the modulator 218 to cause the desired modulation. In other
embodiments, the user may input the pertinent spectral shaping
information directly via the input unit 240.
[0102] FIG. 17 shows the modulation process between the two
excitation masks in more details. The broad-band pulse 250 on the
polarization pulse shaper is s-polarized (0.degree.). After passing
the pulse-shaper, the polarization of every spectral component can
be adjusted to be anywhere between s (0.degree.) and p (90.degree.)
as shown at 252. The analyzer after the electro-optic modulator is
also set to s-polarization. Thus if the electro-optic modulator
does not add any retardance, only the s-polarized portion of the
shaped spectral components 254 are transmitted into the
microscope.
[0103] When the modulator rotates the polarization of the
transmitted beam by 90.degree., only the p-polarized portion of
each spectral component (i.e., the complementary components of the
broadband pulse) is transmitted into the microscope. It is
therefore possible to switch between the selected excitation mask
and it's complimentary at the rate of the electro-optic modulator.
As shown at 256, 258, 260 and 262, alternating between the two
masks provides a complex frequency modulation. Although FIG. 17
shows modulation rate half of the repetition rate of the laser.
Also, FIG. 17 shows the possibility to select either s- or
p-polarization only, any polarization setting possible.
Additionally an amplitude-and-polarization-pulse-shaper can be
used. This allows switching between two completely arbitrary masks,
thus minimizing the power on the sample by blocking unneeded
frequency components to generate the signal for the target
molecule.
[0104] The described approach in FIGS. 16 and 17 shows how
modulation phase can be utilized to detect the pure signal from one
species in the presence of interfering species. The intensities of
mask 1 and mask 2 are modulated exactly 180.degree. out of phase
with respect to each other, which the lock-in detector interprets
as a negative sign and conducts the subtraction of the two masks.
Generalizing this concept, two masks could be modulated 90.degree.
out of phase, which would allow separation of the two channels with
a phase-sensitive detector such as a lock-in amplifier. This would
allow simultaneous two-color imaging with a single lock-in
amplifier, where mask 1 could be read from the x-channel and mask 2
from the y-channel. Alternatively two or even more masks may be
modulated at different modulation frequencies and electronic
processing units looking at these different rates could extract the
individual spectral contributions of the different mask from the
overall detected SRS or CARS from a single detector. In summary,
spectral-temporal modulation of the broadband excitation beam,
allows for encoding of the SRS signal in frequency-domain which can
be analyzed electronically to isolate the individual spectral
contributions.
[0105] Although the above discussion is directed to an application
involving SRS microscopy, the ideas are valid for any type of
contrast in microscopy that is based on excitation spectroscopy
such as CARS, one- and two-photon absorption and emission,
stimulated emission, photo-thermal scattering and photo-acoustic
scattering.
[0106] It can also be applied to fast optical sensing (not
necessarily microscopy) as needed in flow-cytometry. It is also
possible to use a femtosecond-femtosecond configuration (i.e., both
pump and Stokes beam are broadband), for which one or even both
beams are shaped. The excitation masks are not as obvious in this
situation, as all frequency combinations between the two pulses
need to be considered, but they can be determined as the spectral
resolution is solely determined by the spectral resolution of the
pulse-shaper and not the bandwidth of the lasers.
[0107] The determination of the pulse shaping by the controller 242
follows the schemes of chemometrix. Again, with reference to FIG.
7, molecule A (shown at 75) is the analyte of interest, while
elements B (shown at 76) and C (shown at 77) are the interferent
species. Note that the corresponding known Raman spectra
.sigma.(.DELTA..omega.) of A, B, and C are partially overlapping
with each other as shown.
[0108] The objective is to design positive excitation spectral
shapes. In a mixture of A, B and C with unknown concentrations c of
each one, two positive excitation spectral shapes
I.sub.+(.DELTA..omega.) and I.sub.-(.DELTA..omega.) (i.e. masks)
may be designed such that the difference signal .DELTA.S from these
two excitation masks can selectively predict the concentration of
molecule A without getting interference from molecules B and C. For
a given excitation spectral shape I(.DELTA..omega.), the obtained
absorption signal S may be described as discussed above.
[0109] In accordance with certain embodiments, therefore, the
spectral shaper (e.g., a spatial light modulator), may be set to
provide a first mask having a first polarization at the same time
that the spectral shaper is set to provide a second mask having a
second polarization. The spectral shaper, therefore, provides two
polarization distinct masks at the same time without changing. A
polarization modulator may then switch between the two masks very
quickly, permitting real-time subtraction of the results obtained
using the second mask from the results obtained using the first
mask.
[0110] It is also possible to use a femtosecond-femtosecond
configuration (i.e., both pump and Stokes beam are broadband), for
which one or even both beams are shaped, as the spectral resolution
is solely determined by the spectral resolution of the pulse-shaper
and not the bandwidth of the lasers. It can also be applied to fast
optical sensing (not necessarily microscopy) as needed in
flow-cytometry.
[0111] FIG. 18A shows at 270 the spontaneous Raman spectra of the
two biochemicals cholesterol (shown at 272) and oleic acid (shown
at 274) that have no isolated Raman vibrations but distinct Raman
signatures. FIG. 18B shows at 280 the SRS spectra for cholesterol
(shown at 282) and oleic acid (shown at 284) calculated for the
laser excitation spectrum (shown at 286) measured by tuning a
narrowband excitation mask across the broadband spectrum. The
excitation mask specific for cholesterol that suppresses the
interfering signal from oleic acid is generated automatically from
the SRS spectra. Positive and negative spectral components are
highlighted as shown at 288 and 289 respectively.
[0112] FIG. 18C shows at 290 a comparison of spectral and
narrowband detection; the concentration of the interfering species
(oleic acid) is increased, keeping the concentration of the target
species (cholesterol) constant. While the narrowband SRS signal as
shown at 292 falsely increases with increasing concentration of the
interfering species, the spectral SRS signal as shown at 294
predicts the target concentration correctly independent of the
concentration of the interferent and shows no increase.
[0113] Narrowband SRS imaging was achieved by applying a
narrow-band mask to the broadband excitation around 2970 cm.sup.-1.
The suppression of the interfering signal may be quantified as
follows. FIG. 18D shows at 300 the noise of a residual signal
relative to the signal for unshaped excitation for oleic acid.
FIGS. 18E-18G show at 310, 312 and 314 that spectral SRS imaging
allows the suppression of interference from multiple chemical
species. In particular, the images show the same area of a mixture
containing protein extract, oleic acid and stearic acid taken for
three excitation masks optimized for protein (shown at 310), oleic
acid (shown at 312) and stearic acid (shown at 314). The imaging
speed was 30 s per frame for a resolution of 512.times.512 pixels.
The scale-bar is 25 .mu.m.
[0114] FIGS. 19A-19H show data and images associated with the
imaging of lipid storage in system of the multicellular organism
Caenorhabditis Elegans (C. Elegans) obtained by a system in
accordance with an embodiment of the invention. In particular, FIG.
19A shows at 320 an SRS spectrum of the biomolecules oleic acid
(shown at 322), stearic acid (shown at 324) and protein (shown at
326) as computed from the spontaneous Raman library using the
measured laser excitation spectrum (shown at 328).
[0115] FIG. 19B shows at 330 spectral masks computed from spectra
at 320 and used for the imaging. In particular, FIG. 19B shows at
332 a mask for oleic acid, shows at 334 a mask for stearic acid,
and shows at 336 a mask for protein. Spectral images taken from the
same area in a section of a C. Elegans applying these spectral
masks are shown in FIGS. 19C-19E for protein (shown at 340), oleic
acid (shown at 342), and stearic acid (shown at 344). A comparison
of the image for oleic acid (342) and stearic acid (344) shows that
oleic and stearic acid depots co-localize and that there are no
isolated depots of either one or the other. Also, comparison of
oleic acid (342) and protein (340) shows that the lipid depots
further localize with protein-dense organelles.
[0116] FIGS. 19F-19H also show spectral images of protein (shown at
346) and lipid distribution (shown at 348) as well as their overlay
(shown at 350). The arrows shown in image 348 highlight both the
sub-dermal and intestinal lipid storage depots. The imaging speed
was 30 s per frame for a resolution of 512.times.512 pixels. The
scale-bars are 25 .mu.m.
[0117] FIGS. 20A-20L show the label-free microscopy of absorbing
samples. In particular, FIG. 20A shows at 360 an energy diagram of
stimulated Raman scattering (SRS) shown at 362 and two-color
two-photon absorption (TPA) shown at 364. The TPA occurs as a
background signal to SRS and vice versa. FIG. 20B shows at 370
excitation spectra, and in particular, shows excitation spectra of
oleic acid (shown at 372) and a two-photon excitable sample (shown
at 374) as determined from the laser excitation spectrum (shown at
374). FIG. 20C shows at 380 spectral masks used to acquire specific
images of fat and two-photon excitable species. In particular, FIG.
20C shows at 382 a mask for fat, and shows at 384 a mask for the
absorbing species.
[0118] FIGS. 20D-20F show spectral images of a chlorophyll rich
algae sample. A Chlorophyll image (shown at 390) and lipid image
(shown at 394) and their overlap (shown at 396) suggest that the
lipid content of algae can be determined even in the presence of
strongly absorbing (fluorescing) chlorophyll.
[0119] As shown in FIGS. 20G-20L, spectral images of two types of
fat tissue (white fat shown at 396, 398, 400 and brown fat shown at
402, 404, 406) were acquired from fresh tissue, without any
staining, fixing or sectioning. The TPA images (shown at 396 and
402) are dominated by hemoglobin absorption and show the
microvasculature. The fat image (shown at 398 and 404) shows the
different morphology of apidocytes in the different types of
tissue. The image overlays (shown at 400 and 406) highlight that
SRS and TPA are complementary techniques and can increase the
information content of label-free microscopy. The imaging speed was
30 s per frame for a resolution of 512.times.512 pixels. The
scale-bar is 25 .mu.m.
[0120] Those skilled in the art will appreciate that numerous
modifications and variations may be made to the above disclosed
embodiments without departing from the spirit and scope of the
claims.
* * * * *